Journal: bioRxiv

Article Title: Primer- and template-independent RNA polymerization by terminal nucleotidyltransferase TENT4B

doi: 10.64898/2026.03.05.709691

Figure Lengend Snippet: A, Extension of a 20-mer RNA substrate oligo (oligo 1) using rTENT4B and ATP analogs demonstrating differential incorporation. (AMP: Adenosine-5’-monophosphate; ADP: Adenosine-5’-diphosphate; ATP: Adenosine-5’-triphosphate. n=3 replicates, one representative shown. B, Utilization of nucleotide diphosphates (NDPs) by rTENT4B. rTENT4B along with a 20-mer RNA-substrate oligo (oligo 2) was incubated in the presence of four canonical NDPs, in RNA-substrate extension assays, and reaction products were resolved using denaturing TBE/Urea-acrylamide gel followed by Sybr gold staining. C , rTENT4B was incubated with four canonical NDPs plus 5’-Cy5-labeled ATP as substrate. NDP incorporation was confirmed via detecting generation of Cy5-labeled RNA chains after 30 min in no RNA-substrate added reactions. D , De novo ADP polymerization using rTENT4B. rTENT4B along with increasing concentrations of ADP was subjected to RNA-substrate extension assay in the absence of an RNA-substrate, and generation of de novo synthesized RNA products were detected using denaturing TBE/Urea-acrylamide gel followed by Sybr gold staining. E , Consumption of ADP by rTENT4B via analysis of Pi yield, in the presence/ absence of inorganic pyrophosphatase (PPA) as a measure of ATP contamination (ADP + Pi), in the presence/absence of an RNA-substrate oligo, compared to that of rTENT4B alone versus rTENT4B plus ATP. n=4 biological replicates. Mean + S.D. shown, 2-way ANOVA, ns- not significant, ****p<0.001.

Article Snippet: ATP- Adenosine-5’-triphosphate (N0450, New England Biolabs) ADP- Adenosine-5’-diphosphate (NU-1198, Jena Bioscience) AMP- Adenosine-5’-monophosphate (A1752, Sigma) GTP- Guanosine-5’-triphosphate (N0450, New England Biolabs) GDP- Guanosine-5’-diphosphate (G7127, Sigma) CTP- Cytidine-5’-triphosphate (N0450, New England Biolabs) CDP- Cytidine-5’-diphosphate (C9755, Sigma) UTP- Uridine-5’-triphosphate (N0450, New England Biolabs) UDP- Uridine-5’-diphosphate (94330, Sigma) Cy5-ATP- γ-(6-Aminohexyl)-ATP-Cy5- (NU-833-CY5, Jena Bioscience) Cy5-GTP- γ-(6-Aminohexyl)-GTP-Cy5- (NU-834-CY5, Jena Bioscience)

Techniques: Incubation, Acrylamide Gel Assay, Staining, Labeling, Synthesized

Journal: bioRxiv

Article Title: Mechanistic insight into the phosphorylation of ERK by MEK

doi: 10.64898/2026.03.13.710243

Figure Lengend Snippet: a , b , pMEK1-catalyzed dephosphorylation of pY-ERK1. In an ATP-, ADP-, or AMP-PNP-containing buffer, or a nucleotide-free buffer, pY-ERK1 was incubated with various concentrations of pMEK1 at 30, after 3 minutes, the phosphorylation of ERK1 T202 and Y204 was evaluated using Western blotting ( a ) and quantified ( b ). c , d , pMEK1-catalyzed dephosphorylation of pTY-ERK1. In an ADP- or AMP-PNP-containing buffer, or a nucleotide-free buffer, pTY-ERK1 was incubated with pMEK1 at 30. At each time point, the phosphorylation of ERK1 T202 and Y204 was evaluated using Western blotting ( c ) and quantified ( d ). e , f , Effect of mutations of the nucleotide-binding pocket of MEK1 on the activity of pMEK1 to catalyze phosphate group transfer from ERK1 Y204 to T202. In a nucleotide-free buffer, pY-ERK1 was incubated with wild-type (WT) or mutant pMEK1 at 30, and then the phosphorylation of ERK1 T202 and Y204 was evaluated using Western blotting ( e ) and quantified ( f ). The gels in ( a ), ( c ) and ( e ) are the results of a representative experiment out of three independent experiments. The data in ( b ), ( d ) and ( f ) represent the mean ± SD of three independent measurements.

Article Snippet: ATP, ADP (Selleck, Cat# S6325, 100 mM stock in H 2 O) and AMP-PNP (Sigma, Cat# A2647, 100 mM stock in H 2 O) were each prepared as a 4× stock in the reaction buffer.

Techniques: De-Phosphorylation Assay, Incubation, Phospho-proteomics, Western Blot, Binding Assay, Activity Assay, Mutagenesis

Journal: bioRxiv

Article Title: Mechanistic insight into the phosphorylation of ERK by MEK

doi: 10.64898/2026.03.13.710243

Figure Lengend Snippet: a , Summary of the dissociation constants ( K d ) for the binding between MEK1 (uMEK1 or pMEK1) and ERK1 (uERK1 or thio-pTY-ERK1) in the absence or presence of ADP or AMP-PNP. The K d values were measured using Isothermal Titration Calorimetry (ITC). b , Cycle of the pMEK1-catalyzed ERK1 phosphorylation reaction. In addition to the canonical sequential mechanism for pMEK1 (where Y204 and T202 are phosphorylated sequentially by ATP), we proposed a relay mechanism to explain the phosphorylation process of ERK1 T202: Y204 is phosphorylated first, its phosphate group is then transferred to T202, and Y204 is phosphorylated again by ATP to generate dual-phosphorylated ERK1.

Article Snippet: ATP, ADP (Selleck, Cat# S6325, 100 mM stock in H 2 O) and AMP-PNP (Sigma, Cat# A2647, 100 mM stock in H 2 O) were each prepared as a 4× stock in the reaction buffer.

Techniques: Binding Assay, Isothermal Titration Calorimetry, Phospho-proteomics

Journal: bioRxiv

Article Title: Mechanistic insight into the phosphorylation of ERK by MEK

doi: 10.64898/2026.03.13.710243

Figure Lengend Snippet: a , Purified unphosphorylated (uERK1), phosphorylated (pY-ERK1 and pTY-ERK1), and thio-phosphorylated ERK1 (thio-pTY-ERK1) were examined by SDS–PAGE and visualized by Coomassie blue staining. b , Comparison of the ATP consumption rate of thio-pTY-ERK1 with those of uERK1, pY-ERK1, and pTY-ERK1. The ATP consumption was measured using an ADP-Glo Kinase Assay. The MBP peptide FFKNIVTPRTPPPSQGK was used as the substrate of ERK1. The data represent the mean ± SD of three independent measurements. c , d , pMEK1-catalyzed dephosphorylation of pTY-ERK1 ( c ) and thio-pTY-ERK1 ( d ) in an ADP-containing buffer. The percentage of ERK1 in different phosphorylation states was determined by LC-MS.

Article Snippet: ATP, ADP (Selleck, Cat# S6325, 100 mM stock in H 2 O) and AMP-PNP (Sigma, Cat# A2647, 100 mM stock in H 2 O) were each prepared as a 4× stock in the reaction buffer.

Techniques: Purification, SDS Page, Staining, Comparison, Kinase Assay, De-Phosphorylation Assay, Phospho-proteomics, Liquid Chromatography with Mass Spectroscopy